# Core Practical 4

Investigate the effect of temperature, pH, enzyme concentration and substrate concentration on the initial rate of enzyme-catalysed reactions.
OBJECTIVES
● To be able to measure the initial rate of enzyme activity
● To understand why measuring the initial rate is important
● To understand the variables that can affect the rate of an enzyme-catalysed reaction and the result of changing each variable
● To be able to calculate the rate of a reaction using the gradient of a line
SAFETY
● Wear eye protection.
● Avoid skin contact with the trypsin solution. If you get it on your skin, wash it off with cold running water and tell your teacher.
● Hot water from the water baths may be a hazard. If a water bath is above 50 °C, take care not to scald yourself.
MATHS SKILLS
● Use ratios, fractions and percentages.
● Plot two variables from experimental or other data.
● Understand that y = mx + c represents a linear relationship.
● Draw and use the slope of a tangent to a curve as a measure of rate of change.
EQUIPMENT
● eye protection
● skimmed milk powder suspension (2%)
● standard protease (trypsin) solution (1%)
● test tubes and holder
● pipettes
● two cuvettes
● distilled water
● water baths or beakers kept at set temperature
● thermometers
● pH buffers at pH 5, 6, 7, 8, 9, 10

DIAGRAM

figure A Using a colorimeter.

PROCEDURE
Milk protein (casein) is broken down by protease enzymes such as trypsin. The opaque white colour of the milk is replaced by a clear solution. Light passes more easily through the final solution, so the reaction can be monitored using a colorimeter (see figure A).
There are four variables which can change in this investigation: temperature, enzyme concentration, pH or substrate concentration. The procedures for each variable are given below. Although you may not complete all of these investigations, you are expected to have an understanding of the method for each one.
CHOOSING A RANGE OF VALUES
Before you begin, you need to select a range of five values for the independent variable being investigated. When choosing the range of values for your independent variable, it is important to consider safety and validity. Values that are very close together are likely to produce very similar results, which may make it difficult to see a pattern. However, if the intervals between values are too large, this can also make it difficult to identify a pattern. If you use the same interval between the values tested, it will be easier to make a comparison between values. It is also essential to think about safety:
consider whether any of the values chosen could present a hazard during the experiment.
Record your values in the spaces below.
Temperature (Use this space to record your selected temperatures.) pH (Use this space to record your selected pH values.)
Choose a range of buffers so you can test different pH values for the solution. You should consider which buffers to use and the intervals between them. Also consider safety and the risks associated with using very high or very low pH solutions.
Enzyme concentration (Use this space to record your dilutions.)

Plan how you will dilute the 1% trypsin stock solution with distilled water to produce additional test solutions of 0.2%, 0.4%, 0.6% and 0.8%. Aim to produce 10 cm3 of each concentration. Substrate concentration (Use this space to record your dilutions.)
Plan how you will dilute the 2% milk solution with distilled water to produce additional test solutions of 0.2%, 0.4%, 0.6% and 0.8%. Aim to produce 10 cm3 of each concentration.
PROCEDURE – PART 1: TEMPERATURE
1. Prepare water baths for your chosen range of temperatures. Use thermometers to ensure each water bath stays at the correct temperature.
2. Take five test tubes and add 2 cm3 of 1% trypsin solution to each one. Label the tubes with the temperatures and place each one in the corresponding water bath.
3. Take five clean test tubes and add 2 cm3 of milk to each one. Label the tubes with the temperatures and place each one in the corresponding water bath.
4. Leave the test tubes for 5 minutes so they reach the required temperature.
5. Place 2 cm3 of trypsin solution and 2 cm3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero.
6. Pour 2 cm3 of milk suspension at the first temperature being tested into the second cuvette.
7. Add 2 cm3 of trypsin solution from the same water bath to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk by shaking gently, then place the solution into the colorimeter and start the datalogger.
8. Measure the absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance.
9. Rinse the cuvette with distilled water and repeat steps 6–8 for each of the other four temperatures. Remember to use the reference cuvette to zero the colorimeter before each new set of readings. Record the data collected in a results table in the space on page 22.
PROCEDURE – PART 2: PH
1. Select the buffer solutions for the pH values being investigated. Place 1 cm3 of trypsin solution, 1 cm3 of buffer solution and 2 cm3 of distilled water into a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero.
2. Add 1 cm3 of trypsin solution and 1 cm3 of your first buffer solution to a cuvette.
3. Measure 2 cm3 of milk suspension into a second cuvette.
4. Add the mixture made in step 2 to the milk in the cuvette. Working quickly, mix the solution and the milk by shaking gently, then place the solution into the colorimeter and start the datalogger.
5. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat steps 2–5 for each of the other four pH values. Remember to use the reference cuvette to zero the colorimeter before each new set of readings. Record the data collected in a results table in the space on page 22.

PROCEDURE – PART 3: ENZYME CONCENTRATION
1. Check your dilution calculations, then make up the solutions as planned.
2. Place 2 cm3 of trypsin solution and 2 cm3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero.
3. Measure 2 cm3 of milk suspension into a second cuvette.
4. Add 2 cm3 of your first trypsin solution to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk, then place the cuvette into the colorimeter and start the datalogger.
5. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat steps 3–5 for each of the other four concentrations. Remember to use the reference cuvette to zero the colorimeter before each new set of readings. Record the data collected in a results table in the space on page 22.
PROCEDURE – PART 4: SUBSTRATE CONCENTRATION
1. Check your dilution calculations, then make up the solutions as planned.
2. Place 2 cm3 of 1% trypsin solution and 2 cm3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero.
3. Measure 2 cm3 of the 0.2% milk suspension into a second cuvette.
4. Add 2 cm3 of trypsin solution to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk, place the solution into the colorimeter and start the datalogger.
5. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat steps 3–5 for each of the other four milk concentrations. Remember to use the reference cuvette to zero the colorimeter before each new set of readings. Record the data collected in a results table in the space on page 22.
ANALYSIS OF RESULTS
1. Plot a graph of absorbance against time. It should be possible to plot each variable as a different line on the same axes.
2. Use your graph to determine the initial rate of reaction for each factor. Do this by drawing a tangent to the initial part of each curve and calculating the gradient of each line.
3. Draw a second graph to show the initial rate of reaction against each factor you changed.
4. Write a short conclusion to describe and explain the results of this investigation
LEARNING TIPS

● Use a sharp pencil when drawing graphs. Use different symbols around plotted points to distinguish lines when several concentrations are plotted on one set of axes. Remember to include a key.
● Keep graph scales simple. Use one large square to represent 5, 10 or 20 (or perhaps 0.05, 0.1 or 0.2) when plotting intermediate points because then the smaller squares will have values that are easy to work with.
● To calculate the initial rate of a reaction, draw a tangent to the line at the steepest point. This line can then be turned into a right-angled triangle. To calculate the gradient of the line, divide the change in the value on the y-axis by the change in the value on the x-axis:

● It is unlikely that you will have time to investigate all four factors. However, you should be aware of the methods used.